CRISPR/Cas12a with Antisense Oligonucleotide-Regulated Translational Amplification for Ultrasensitive Nucleic Acid Detection.

Highly sensitive nucleic acid testing-assisted early disease detection is crucial for effective disease prevention and management, particularly when targeting low-abundance genetic materials in molecular diagnostics. This study describes CRATE (CRISPR/Cas controlled antisense oligonucleotide (ASO)-mediated translational signal enhancement), a novel ultrasensitive approach for nucleic acid detection by integrating Cas12a trans-cleavage, ASO-controlled gene expression, and cell-free signal protein amplification. This assay leverages the target-induced trans-cleavage of ASO-controlled gene expression for the amplification of signal proteins, with luminescent signal allowing for attomolar-level target DNA detection, as well as antigenic protein application enabling visual detection by lateral flow assay. The CRATE assay improves sensitivity using ASO-modified locked nucleic acid, achieving a 10-aM-level DNA detection. The proof of concept demonstrates 0.1 copies/μL detection of HPV genomic DNA from HPV-positive cancer cells as well as colorimetric lateral flow tests with ∼10 copies/μL sensitivity. The CRATE assay can detect the HBV target in plasma from HBV-positive patients with 100% sensitivity and specificity. With high specificity and accuracy, the CRATE assay retains the potential for detecting any nucleic acid of interest. By integration of precise CRISPR-based cleavage, ASO regulation, and efficient protein signal amplification, this approach provides a promising solution for highly selective and sensitive nucleic acid detection and potential applications in clinical diagnostics and point-of-care testing.